ABSTRACT Skeletal muscle is inherently regenerative following acute injury. It is well established that the spatiotemporal dynamics of the responding immune cell populations are critical determinants of the regenerative process. Specifically, an appropriately timed switch from a type-I to a type-II immune response is required for skeletal muscle regeneration following injury, and this self-regenerative capacity is lost after a critical size volumetric muscle loss (VML) event such as trauma or tumor excision. We recently showed that an acellular biologic scaffold composed entirely of extracellular matrix (ECM) can facilitate a macrophage phenotype transition that leads to downstream site-appropriate functional tissue deposition and myogenesis as a treatment for volumetric muscle loss in preclinical animal models and in 13 human patients. Our current objective is to gain translatable mechanistic insights into the immunobiology behind both normal skeletal muscle regeneration following acute injury and in the presence of an ECM bioscaffold with the broad aim of developing therapeutics that enable and direct immune cells to facilitate constructive, functional remodeling after VML. The proposed studies will investigate the ability and necessity of a newly identified component of the ECM, the interleukin-33 (IL-33), to influence remodeling after skeletal muscle injury. Typically found in the nucleus of stromal cells, IL-33 has been shown to be a potent mediator of skeletal muscle, cardiac muscle, lung epithelium, and dermal repair via poorly defined mechanisms involving immune cells expressing the IL-33 receptor, ST2. The subject matter of the present proposal is based upon our discovery that IL-33 is stably integrated into the ECM via encapsulation within matrix bound nanovesicles (MBV) thereby protecting IL-33 from rapid oxidation. Following ECM degradation, MBV are released from the matrix, taken up by immune cells wherein IL-33 activates macrophages towards a pro-remodeling phenotype via a non-canonical ST2-indendent pathway. The discovery of IL-33 as an integral component of ECM-MBV represents a distinct therapeutic target and marker of tissue remodeling. Furthermore, our experimental design will allow for the first in-depth molecular characterization of the genes and signaling pathways regulated by the ST2-independent IL-33 pathway and will greatly advance our understanding of the molecular mechanisms by which ECM facilitates the functional remodeling response. Separately, the use of ECM therapies (either as a hydrogel as is presently being tested in a Phase I clinical trial by Ventrix for cardiac repair) or as a bioscaffold sheet (recently used as a treatment for VML in a 13 patient cohort study) can now be studied from a new perspective, and will help guide the design of next generation products, diagnostics and therapeutic applications.